Table S1: Sequences of the three PCR primer pairs A, B, and C applied to
determine the CEBPA mutational status.
Gene
Nucleotide sequence
CEBPA-A-F
CEBPA-A-R
CEBPA-B-F
CEBPA-B-R
CEBPA-C-F
CEBPA-C-R
5'-TCG CCA TGC CGG GAG AAC TCT AAC-3'
5'-AGC TGC TTG GCT TCA TCC TCC T-3'
5'-CCG CTG GTG ATC AAG CAG GA-3'
5'-CCG GTA CTC GTT GCT GTT CT-3'
5'-CAA GGC CAA GAA GTC GGT GGA CA-3'
5'-CAC GGT CTG GGC AAG CCT CGA GAT-3'
Table S2A: Molecular characterization of CEBPA mutations.
# pat. age FAB N-mutation
Single CEBPA mutations:
1
38 M2 236-237insGC
2
51 M2 392-393insT
3
55 M2 245delG
4
49 M1 216-217insCG
5
22 M1
6*
33 M2
7”
53 M1
C-mutation
1079-1080insTCT
Double CEBPA mutations:
1
40 M2 563-564insCG
1094-1095insCTG
2
57
M2 327-328insC
3
52
M2 395delC
4
47
M1 213delC
1098-1099insGTC
1076-1077insAAG
1088-1089insTCT
5
44
M2 327-328insC
1098-1099insGTC
6
27
M2 319insT
7
40
M1 291delC
940insAAG
937insCAG
8
52
M2 286-287insTC
1076-1077insCCG
9
51
M2 327insT
1094-1095insCTG
10
53
M1 291insGC
11
39
M1 213insAG
1076-1077insACG
1088-1089insCCG
12
29
M1 395insCG
937insCAG
others
AA change
A29fsX160
A91fsX107
G32fsX159
P23fsX160
S310-311ins
692C>G Y181X
1083C>T Q312X
Y138fsX160
L315-316ins
E59fsX107
V316-317ins
F82fsX159
K309-310ins
P22fsX159
S313-314ins
E59fsX107
V316-317ins
D106X
314insK
A47fsX159
313insQ
P46fsX160
K309-310ins
E59fsX107
L315-316ins
A47fsX160
K309-310ins
P22fsX160
S313-314ins
F82fsX160
313insQ
One patient had the following two point mutations of unknown significance and
was not considered in this analysis: 1167G>A (inducing G340S) and 744745GC>TT (inducing A199L). *This patient also had the following point
mutations: 672C>G, 676C>T, 678-679GG>TT, and 683C>T; they were all
located on the same allele also carrying the 692C>G (encoding a novel stop
codon) as identified by allele specific cloning of PCR products. * and “: These two
patients had point mutations encoding a novel stop codon downstream of the
ATG located at amino acid position 120. Thus, formation of the wild-type 42kDa
and of the 30ka peptides is both affected.
Table S2B: Clinical course of AML patients with CEBPA mutations.
# pat. Age FAB
DFS
(months)
OS
(months)
Single CEBPA mutations:
1
38 M2
6
2
51 M2
32
3
55 M2
12
4
49 M1
1
5
22 M1
22
6*
33 M2
42
7”
53 M1
3
8
33
15
3
23
43
5
Double CEBPA mutations:
1
40 M2
40
41
follow-up
(months)
relapse
(months)
AA change
yes
no
yes
yes
no
no
yes
A29fsX160
A91fsX107
G32fsX159
P23fsX160
S310-311ins
Y181X
Q312X
41
no
28
no
Y138fsX160
L315-316ins
E59fsX107
V316-317ins
F82fsX159
K309-310ins
P22fsX159
S313-314ins
E59fsX107
V316-317ins
D106X
314insK
A47fsX159
313insQ
P46fsX160
K309-310ins
E59fsX107
L315-316ins
A47fsX160
K309-310ins
P22fsX160
S313-314ins
F82fsX160
313insQ
33
23
43
2
57
M2
23
28
3
52
M2
2
3
4
47
M1
33
34
34
no
5
44
M2
35
36
36
no
6
27
M2
15
16
16
no
7
40
M1
33
34
34
no
8
52
M2
28
32
32
no
9
51
M2
11
12
10
53
M1
41
42
11
39
M1
20
24
12
29
M1
37
38
yes
yes
42
no
yes
38
no
DFS: disease-free survival; OS: overall survival; Follow-up in months is indicated
for patients alive and disease-free from time of diagnosis censored at their last
follow-up visit.
Table S3: Clinical characteristics of AML patients according to CEBPA
mutational status.
all
CEBPA
CEBPA
wt
CEBPA
CEBPA
mut
single
double
(n=224)
(n=205)
(n=19)
(n=7)
(n=12)
______________________________________________________________________
_
sex
102f/112m 93f/102
9f/10m
3f/4m
6f/6m
median age*
53
54
51
53
49
median WBC G/L
19.8
27.8
12.1
14.1
11.2
(range)
(0.6-360) (0.6-360)
(2.8-36)
(3.2-36)
(2.828)
median % blasts in blood
68
72
60
65
58
(range)
(0-99)
(0-99)
(12-90)
(22-90)
(12-88)
median LDH units/L°
808
848
515
535
492
extramedullary
manifestations (n)
51
48
3
1
2
de novo AML, n
204
194 19
7
12
secondary AML, n
20
20
0
0
0
MDS (n)
17
17
0
0
0
therapy-related (n)
3
0
0
0
0
consolidation in CR1+
chemotherapy
(n)
autologous transplant (n)
allogenous transplant (n)
FAB classification:
M0
M1
M2
M4
M5
M6
M7
72
54
33
54
34
33
9
10
0
3
4
0
6
6
0
20
61
56
48
31
5
3
20
53
47
48
31
5
3
0
8
11
0
0
0
0
0
3
4
0
0
0
0
0
5
7
0
0
0
0
*All patients were younger than 61 years at diagnosis. °LDH normal <480 units/L. +159
of 224 patients achieved a first complete remission after two cycles of induction
chemotherapy and thus underwent consolidation therapy. P-values were calculated with
the Mann-Whitney test. No significance of p<0.05 was achieved for any comparison
between single versus double CEBPA mutation groups. Significant differences were
observed between CEBPA mutant and CEBPA wild-type patients for WBC at diagnosis
(p=0.012), for LDH at diagnosis (p=0.032), and for allogenous transplant as
consolidation treatment in CR1 (p=0.003). Abbreviations: FAB, French American British
classification; WBC, white blood cell count; LDH, lactate dehydrogenase. MDS,
myelodysplastic syndrome.
Table S4: Molecular and karyotype abnormalities of AML patients
according to CEBPA mutational status.
all
(n=224)
CEBPA
CEBPA
wt
(n=205)
CEBPA
CEBPA
mut
(n=19)
single
(n=7)
double
(n=12)
______________________________________________________________________
_
FLT3-ITD n(%)
NPM1 mutation n(%)
63(28)
102(46)
62
101
good-risk* n(%)
intermediate-risk” n(%)
bad-risk+ n(%)
38(17)
121(54)
65(29)
38
104
63
1
1
1
1
0
17
2
0
0
0
6
1°
0
11
1#
*Good-risk patients comprised t(8;21) and inv(16) with n=20, and n=18 patients,
respectively. “Intermediate-risk patients showed a normal karyotype (n=111), +8 (n=8),
and -Y (n=2). +Bad-risk patients comprised all other karyotype results. °This patient had
monosomy 7. #This patient had del6q24. AML patients with CEBPA mutations had
significantly less FLT3-ITD (p=0.023) and NPM1 mutations (p=0.008).
Table S5: Clinical outcome of AML patients according to CEBPA mutational
status.
all
(n=224)
CEBPA
CEBPA
wt
(n=205)
CEBPA
CEBPA
mut
(n=19)
single
(n=7)
double
(n=12)
______________________________________________________________________
_
CR1 achieved, n(%)
Death in CR1, n
Relapse, n(%)
of patients with CR1
OS at two years (%)
DFS at two years (%)
Patients disease-free
in follow-up, n(%)
median, months
159(71)
10
140
10
19
0
137(61)
48
46
130(63)
46
44
7(37)
69
63
4(54)
43
43
12
31
3
33
83(37)
37
76
38
7
0
12
0
3(25)
83
75
9
34
Abbreviations: first complete remission, CR1; overall survival, OS; disease-free survival,
DFS. At two years, OS and DFS were significantly shorter in AML with single versus
double CEBPA mutations (p=0.005, and p=0.011, respectively).